Project Summary/Abstract Conjugated proteins are an essential tool for biomedical research. Techniques like immunohistochemistry and flow cytometry rely completely on fluorophore-labeled antibodies, and antibody?drug conjugates are increasingly used as immunotherapies. For many applications, the final mole ratio of label-to-protein, or labeling ratio, must be precisely controlled to maximize the effect of the label while preserving the function of the protein. With the increasing requirement for reagent validation, optimization of labeling ratio should be performed prior to validation. Traditionally, each labeling reaction requires at least 30-50 g of precious and often expensive protein, making optimization experiments a costly endeavor. Furthermore, existing microfluidic devices to measure protein labeling rely on specialized equipment such as syringe pumps, pressure systems, and supplies for electrophoretic separation, which may not be readily available to biomedical labs who just want to label their protein efficiently. The goal of this focused R03 project is to enable easy optimization of protein conjugation by developing a rapid, small scale, multiplexed analysis of labeling ratio on a handheld microfluidic SlipChip device. The chip will be designed to require no pumps nor a power supply to operate: only hands and a pipet. Readout will be on a conventional plate reader, which is ubiquitous in biomedical research labs. The chip will consume at least 10-fold less protein per reaction than traditional methods and will test five fluorescent dye:protein reagent ratios in parallel. It will be validated to ensure that its predictions can be scaled up for practical lab use. In preliminary work, we designed, fabricated, and individually tested the three functional modules of the device: dilution of dye, reacting dye and protein sample, and removing un-reacted dye by size exclusion. Here, we will refine and integrate the current modules into a single fully functional handheld chip that consumes less than 5 g of protein sample (AIM 1). We will then move to a 3D printed design for moderate-throughput production and integration of a 3D printed guide for slipping, after identifying a suitable transparent resin (AIM 2). 3D printing will enable production and future dissemination to other research labs, eliminate the use of bulky clips on the device, and facilitate accurate slipping. To validate the accuracy of the assay, we will test multiple proteins and dyes in labeling reactions on-chip and compare them to full-scale reactions. If successful, this research will generate a user-friendly device for rapid and multiplexed optimization of protein labeling ratio. We envision that the impact of this innovative chip will be high, by facilitating the optimization of reagents prior to validation. This will improve the quality of reagents used for biomedical research, diagnostics, and therapeutics. The work will simultaneously advance the field of microfabrication by creating a road map for conversion from glass etching to 3D printing with unique design features.